MicroRNAs (miRNAs) regulate many biological processes, and altered miRNA expression or function is associated with human diseases including a variety of cancers, heart disease, and diabetes. However, the molecular mechanisms by which specific miRNAs regulate complex cellular and organismal functions is poorly understood, largely because very few direct targets of miRNAs have been experimentally identified. Predictions of miRNA targets by computational methods are widely variable and not yet reliable. In this proposal, I describe a novel biochemical method for the systematic and direct identification miRNA targets. Through biochemical isolation of an RNP complex, shown to be required for miRNA-mediated gene silencing, that contains both miRNAs and their actively repressed miRNA targets, I will simultaneously identify bona fide miRNA targets and monitor the dynamic expression of miRNAs. This methodology will be employed in vivo in Caenorhabditis elegans to identify miRNA targets in specific tissues, during development, and in response to organismal stresses to address the hypothesis that miRNA-mediated gene regulation is highly dynamic and tightly regulated. The studies in this proposal are intended to achieve two major goals: 1) to identify miRNAs and their targets that mediate key developmental decisions and organismal stress responses, and 2) to refine our understanding of the mechanisms that direct miRNA target gene selection. The data generated in these studies will provide insight into the mechanisms by which miRNAs regulate processes such as proliferation, differentiation, and stress responses, and how miRNA dysfunction can lead to disease states. This proposal seeks to identify mechanisms of post-transcriptional gene regulation that mediate important cellular functions such as proliferation and differentiation. Disruption of these mechanisms of gene regulation is associated with human diseases such as cancer, heart disease, and diabetes.